Catalytic methods for selective oxidation of carbon-hydrogen (C—H) bonds are of huge synthetic utility, as they can simplify the synthesis of complex molecules, providing more concise, atom-economical, and convenient routes for the preparation and derivatization of these compounds. In the context of bioactive molecules, for example, site-selective introduction of oxygenated functionalities at unreactive C—H positions can itself contribute to improve the physico-chemical, pharmacokinetic or pharmacological properties of these compounds. Additionally, these transformations can facilitate further functional elaboration of these compounds through interconversion of the newly installed oxygenated functionalities into other functional groups in order to obtain more potent or bioavailable derivatives.
Selective oxidation of C—H bonds and in particular aliphatic C—H bonds is a difficult chemical transformation due to the chemical inertness of these bonds towards most chemical reagents, the abundance of C—H bonds in synthetic and naturally occurring molecules, and the higher reactivity of the oxidized products compared to the reagents, which can lead to undesired overoxidation reactions (Gunay, A.; Theopold, K. H. Chem. Rev., 2010, 110, 1060; Stahl, S. S.; Labinger, J. A.; Bercaw, J. E. Angew. Chem. Int. Ed. Engl., 1998, 37, 2181; Punniyamurthy, T.; Velusamy, S.; Iqbal, J. Chem. Rev., 2005, 105, 2329). This makes the development of efficient oxidation catalysts that possess sufficient chemical reactivity while maintaining high chemo-, regio-, and stereoselectivity a considerable challenge. Considerable effort has been devoted over the past decades towards the development of chemical methods for selective oxidation of aliphatic C—H bonds. These involve the use of oxidizing reagents (Brodsky, B. H.; Du Bois, J. J Am Chem Soc, 2005, 127, 15391; Wender, P. A.; Hilinski, M. K.; Mayweg, A. V. Org. Lett., 2005, 7, 79; Lee, S.; Fuchs, P. L. J Am Chem Soc, 2002, 124, 13978; Gomez, L.; Garcia-Bosch, I.; Company, A.; Benet-Buchholz, J.; Polo, A.; Sala, X.; Ribas, X. Angew. Chem. Int. Ed. Engl., 2009, 48, 5720; Chen, K.; Baran, P. S, Nature, 2009, 459, 824), supramolecular catalysts (Grieco, P. A.; Stuk, T. L. J Am Chem Soc, 1990, 112, 7799; Cook, B. R.; Reinert, T. J.; Suslick, K. S. J Am Chem Soc, 1986, 108, 7281; Yang, J.; Gabriele, B.; Belvedere, S.; Huang, Y.; Breslow, R. J. Org. Chem., 2002, 67, 5057; Das, S.; Incarvito, C. D.; Crabtree, R. H.; Brudvig, G. W. Science, 2006, 312, 1941; Das, S.; Brudvig, G. W.; Crabtree, R. H. Chem Commun, 2008, 413), biomimetic catalysts (Mahadevan, V.; Gebbink, R. J. M. K.; Stack, T. D. P. Curr. Opin. Chem. Biol., 2000, 4, 228; Que, L., Jr.; Tolman, W. B. Nature, 2008, 455, 333), and organometallic catalysts (Chen, M. S.; White, M. C. Science, 2007, 318, 783; Chen, M. S.; White, M. C. Science, 2010, 327, 566; Dick, A. R.; Hull, K. L.; Sanford, M. S. J Am Chem Soc, 2004, 126, 2300; Dick, A. R.; Sanford, M. S. Tetrahedron, 2006, 62, 2439). Despite this progress, these methods suffer from a number of drawbacks, such as having limited catalytic efficiency (i.e., low turnover numbers), requiring the presence of ‘directing groups’ pre-installed in the target molecule, and/or allowing selective targeting of only electronically activated (e.g., tertiary or heteroatom-bearing) C—H sites in the molecule of interest. Moreover, such oxidation reagents/catalysts are not readily amenable to modulation of the regio- and stereoselectivity in order to target different C—H sites in the same target molecule.
While selective oxyfunctionalization of organic molecules via chemical methods remains difficult, several enzymatic systems occur in nature that are able to carry out this transformation under mild reaction conditions such as ambient pressure and temperature and in aqueous solvents. Monooxygenases (EC 1.13 and EC 1.14) are an important class of enzymes that catalyze the insertion of a single oxygen atom from molecular oxygen (O2) into the aliphatic or aromatic C—H bond of an organic substrate (Tones Pazmino, D. E.; Winkler, M.; Glieder, A.; Fraaije, M. W. Journal of biotechnology, 2010, 146, 9; Lewis, J. C.; Coelho, P. S.; Arnold, F. H. Chem Soc Rev, 2010). Monooxygenases are classified according to the enzyme-bound cofactor involved in oxygen activation and include heme-dependent, flavin-dependent, copper-dependent, non-heme iron-dependent, pterin-dependent, and cofactor-independent monooxygenases (Tones Pazmino, D. E.; Winkler, M.; Glieder, A.; Fraaije, M. W. Journal of biotechnology, 2010, 146, 9; Lewis, J. C.; Coelho, P. S.; Arnold, F. H. Chem Soc Rev, 2010).
Heme-dependent monooxygenases, also referred to as cytochrome P450 monooxygenases or CYPs are a large class of enzymes found in both eukaryotic and prokaryotic organisms, including bacteria, fungi, plants, insects, and mammals. Cytochrome P450 enzymes are defined by the presence of a heme (iron protoporphyrin IX) prosthetic group coordinated on the proximal side by a thiolate ion of a conserved cysteine residue (Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem. Rev., 2005, 105, 2253; Ortiz de Montellano, P. R. Chem. Rev., 2010, 110, 932). The typical reaction catalyzed by P450 enzymes involves the reductive activation of molecular oxygen, using electrons equivalents derived from reduced pyridine nucleotides (NADH or NADPH), and subsequent insertion of one of the oxygen atoms into the substrate with concomitant reduction of the second oxygen atom to water (Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem. Rev., 2005, 105, 2253; Ortiz de Montellano, P. R. Chem. Rev., 2010, 110, 932). Depending on the electron transport systems, cytochrome P450 enzymes have been divided in several classes (e.g., class I, class II, class III, class IV) albeit this way of classification have changed over time as new typologies of P450 systems have been discovered (Bernhardt, R. Journal of biotechnology, 2006, 124, 128). Cytochrome P450s can also operate as peroxidases utilizing hydrogen peroxide as oxidant species and the peroxide shunt pathway in the catalytic cycle without the need of a NAD(P)H-oxidizing redox partner (Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem. Rev., 2005, 105, 2253). In addition to hydroxylation, P450 monooxygenases are also capable of catalyzing other reactions, including epoxidation, heteroatom (e.g., N, S, O) dealkylation, heteroatom (e.g., N, S) oxidation, oxidative deamination, dehydrogenation, dehydration, oxidative C—C bond cleavage, and rearrangements (Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. Rev., 1996, 96, 2841; Guengerich, F. P. Curr Drug Metab, 2001, 2, 93). More than 10,000 distinct members of the P450 family have been identified to date. Natural P450 enzymes are known to catalyze the oxidation of a wide range of structurally diverse substrates, including fatty acids, drugs, steroids, and numerous other natural products and small molecules. Despite their broad substrate and reaction scope and sequence identities as low as 20%, members of the P450 family share a conserved structural fold as revealed by comparison of the crystal structure of P450s isolated from bacterial, mammalian, and plant sources (Pylypenko, O.; Schlichting, I. Annual review of biochemistry, 2004, 73, 991; Graham, S. E.; Peterson, J. A. Arch. Biochem. Biophys., 1999, 369, 24).
Another class of synthetically and biotechnologically valuable monooxygenase enzymes are flavin-dependent monooxygenases (Tones Pazmino, D. E.; Winkler, M.; Glieder, A.; Fraaije, M. W. Journal of biotechnology, 2010, 146, 9; van Berkel, W. J. H.; Kamerbeek, N. M.; Fraaije, M. W. Journal of biotechnology, 2006, 124, 670). Flavin-dependent monooxygenases are able to catalyze numerous reactions, including C—H bond hydroxylations, epoxidations, Bayer-Villiger oxidations, and sulfoxidations (Tones Pazmino, D. E.; Winkler, M.; Glieder, A.; Fraaije, M. W. Journal of biotechnology, 2010, 146, 9; van Berkel, W. J. H.; Kamerbeek, N. M.; Fraaije, M. W. Journal of biotechnology, 2006, 124, 670). The flavins associated to these enzymes, either covalently or non-covalently, are either FMN or FAD. According to their amino acid similarities, members of the flavin-dependent monooxygenases family have been subdivided into six classes, namely A, B, C, D, E, and F (Tones Pazmino, D. E.; Winkler, M.; Glieder, A.; Fraaije, M. W. Journal of biotechnology, 2010, 146, 9; van Berkel, W. J. H.; Kamerbeek, N. M.; Fraaije, M. W. Journal of biotechnology, 2006, 124, 670). To perform the oxidation reactions, these enzymes generate a reactive intermediate upon reaction between molecular oxygen and the reduced enzyme-bound flavin. Depending on the protonation state, the reactive intermediate causes the monooxygenation of the substrate via a nucleophilic or an electrophilic mechanism. In most cases, reduction of the enzyme-bound flavin is achieved through oxidation of reduced coenzymes NADPH or NADH. Examples exist, however, where the flavin is reduced by the substrate itself.
The ability of monooxygenases to catalyze, among other reactions, the oxidation of aromatic and aliphatic C—H bonds with high catalytic efficiency and under mild reaction conditions makes them attractive platforms for the development of biocatalysts for selective oxidation of organic molecules (Fasan, R.; Chen, M. M.; Crook, N. C.; Arnold, F. H. Angew. Chem. Int. Ed. Engl., 2007, 46, 8414; Lewis, J. C.; Bastian, S.; Bennett, C. S.; Fu, Y.; Mitsuda, Y.; Chen, M. M.; Greenberg, W. A. Proc. Natl. Acad. Sci. USA, 2009, 106, 16550; Li, S.; Chaulagain, M. R.; Knauff, A. R.; Podust, L. M.; Montgomery, J.; Sherman, D. H. Proc. Natl. Acad. Sci. USA, 2009, 106, 18463; Whitehouse, C. J.; Bell, S. G.; Tufton, H. G.; Kenny, R. J.; Ogilvie, L. C.; Wong, L. L. Chem Commun, 2008, 966; Zehentgruber, D.; Hannemann, F.; Bleif, S.; Bernhardt, R.; Lutz, S. Chembiochem, 2010, 11, 713; Sun, L.; Chen, C. S.; Waxman, D. J.; Liu, H.; Halpert, J. R.; Kumar, S. Arch. Biochem. Biophys., 2007, 458, 167; Liu, L.; Schmid, R. D.; Urlacher, V. B. Biotechnol. Lett., 2010, 32, 841; Bottner, B.; Schrauber, H.; Bernhardt, R. J. Biol. Chem., 1996, 271, 8028; Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. H. J Am Chem Soc, 2003, 125, 13442; Tang, W. L.; Li, Z.; Zhao, H. Chem Commun, 2010, 46, 5461). In addition to being inherently ‘green’ and inexpensive to produce, these biological oxidation catalysts offers the advantage that their regio- and stereoselectivity can be modulated by protein engineering and potentially directed also towards energetically and/or stereoelectronically unactivated aliphatic and aromatic C—H bonds. Recently, the systematic utilization of P450 variants with diversified substrate profile and regioselectivity has constituted a powerful strategy towards the late-stage transformation of single and multiple unactivated sp3 C—H bonds in small-molecule substrates through P450-mediated chemoenzymatic synthesis (Rentmeister, A.; Arnold, F. H.; Fasan, R. Nat Chem Biol, 2009, 5, 26).
While many different monooxygenases can be isolated from natural sources or produced by protein engineering, a bottleneck remains, namely the time and screening effort required to identify the variant(s) with the suitable level of catalytic activity (i.e., turnover number and turnover rate) and selectivity (i.e., chemo-, regio- and stereoselectivity) for the intended synthetic application. Typically, this requires the screening of large libraries of natural or engineered monooxygenases by GC- or HPLC-based methods, which are inherently low throughput and involve extensive sample manipulation. The use of fluoro/chromogenic substrate surrogates can accelerate this process (Fasan, R.; Chen, M. M.; Crook, N. C.; Arnold, F. H. Angew. Chem. Int. Ed. Engl., 2007, 46, 8414; Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold, F. H. J Am Chem Soc, 2003, 125, 13442; Fasan, R.; Meharenna, Y. T.; Snow, C. D.; Poulos, T. L.; Arnold, F. H. J. Mol. Biol., 2008, 383, 1069; Schwaneberg, U.; Schmidt-Dannert, C.; Schmitt, J.; Schmid, R. D. Anal. Bioanal. Chem., 1999, 269, 359; Ghosal, A.; Hapangama, N.; Yuan, Y.; Lu, X.; Horne, D.; Patrick, J. E.; Zbaida, S. Biopharm. Drug. Dispos., 2003, 24, 375), but these methods are limited in scope in that they are useful only in the context of a single target substrate. Furthermore, none of the methods currently available for high throughput screening of monooxygenase activity (e.g., Rabe, K. S.; Gandubert, V. J.; Spengler, M.; Erkelenz, M.; Niemeyer, C. M. Anal. Bioanal. Chem., 2008, 392, 1059) provides qualitative or quantitative information, regarding the regio/stereoselectivity of the screened enzymes, which has to be established on a case-by-case basis through laborious and time-consuming HPLC or GC analyses.
Molecular modeling methods have been proposed for predicting substrate binding to monooxygenase enzymes such as, for example, P450 enzymes (Stjernschantz, E.; Oostenbrink, C. Biophys. J., 2010, 98, 2682; Harris, D. L.; Park, J. Y.; Gruenke, L.; Waskell, L. Proteins, 2004, 55, 895; Terfloth, L.; Bienfait, B.; Gasteiger, J. J. Chem. Inf. Model., 2007, 47, 1688; Vasanthanathan, P.; Olsen, L.; Jorgensen, F. S.; Vermeulen, N. P.; Oostenbrink, C. Drug Metab. Dispos., 2010, 38, 1347). However, the plasticity and dynamic nature of these enzymes hampers the formulation of accurate and reliable predictions (Hritz, J.; de Ruiter, A.; Oostenbrink, C. J. Med. Chem., 2008, 51, 7469; Ekroos, M.; Sjogren, T. Proc. Natl. Acad. Sci. USA, 2006, 103, 13682). In addition, these methods do not discriminate between inhibitors and substrates (Stjernschantz, E.; Oostenbrink, C. Biophys. J., 2010, 98, 2682; Vasanthanathan, P.; Olsen, L.; Jorgensen, F. S.; Vermeulen, N. P.; Oostenbrink, C. Drug Metab. Dispos., 2010, 38, 1347). Finally, these approaches require prior knowledge of the enzyme structure which may not be available or readily available, in particular when a multitude of different monooxygenases, either natural or engineered, are to be evaluated.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.